Interleukin-8 and Monocyte Chemotactic Protein-1 Gene Expression and Protein Production by Human Orbital Fibroblasts

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Abstract

Orbital inflammation is common, but the mechanisms underlying leukocytic infiltration of orbital tissue are poorly understood. We studied resident human orbital fibroblasts (OF) interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) mRNA expression and protein secretion in response to lipopolysaccharide (LPS) or recombinant human cytokines that are present during inflammation.

Third-passaged cultured human OF were left unstimulated or incubated with varying concentrations of LPS, recombinant interleukin-1-beta (rIL-1β), recombinant tumor necrosis factor-alpha (rTNF-α), or recombinant interferon-gamma (rIFN±γ) for 2, 4, 8, or 24 h. Northern blot analysis and ELISA were performed to determine OFIL-8 and MCP-1 mRNA expression and protein secretion, respectively. Experiments were performed in triplicate and repeated four times on different cell lines.

OF lacked constitutive IL-8 or MCP-1 gene expression, but produced substantial dose-dependent increases in steady-state IL-8 and MCP-1 mRNA expression by 2 h of LPS or cytolkine stimulation (rIL-lβ>rTNF-α >LPS>rIFN-γ), maintained at 24 h. ELISA for IL-8 and MCP-1 proteins showed significant time- and dose-dependent OF secretion after exposure to recombinant cytokine or LPS (rIL-lβ>rTNF-α>LPS), measured after 4 h of exposure (p < 0.01). This increased in the media over the next 20 h. rIFN-γ was a potent stimulant of OF MCP-1, significant by 2 h (p < 0.05), but only a weak stimulant of IL-8 at 24 h.

OF secreted IL-8 and MCP-1 in response to LPS and proinflammatory cytokines, indicating that these resident cells within the orbit have the capacity to actively participate in the initiation and propagation of orbital inflammation. Strategies aimed at modulating local mediators may be helpful in the management of orbital inflammatory disease.

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